Gluconeogenesis in the yolk syncytial layer‐like tissue of cloudy catshark (Scyliorhinus torazame)

Abstract Glucose has important roles in the development of zebrafish, the vertebrate animal model; however, in most oviparous animals, the amount of maternally provided glucose in the yolk is scarce. For these reasons, developing animals need some ways to supplement glucose. Recently, it was found that developing zebrafish, a teleost fish, undergo gluconeogenesis in the yolk syncytial layer (YSL), an extraembryonic tissue that surrounds the yolk. However, teleost YSL is evolutionarily unique, and it is not clear how other vertebrates supplement glucose. In this study, we used cloudy catshark (or Torazame catshark), an elasmobranch species which possesses a YSL‐like tissue during development, and sought for possible gluconeogenic activities in this tissue. In their yolk sac, glucose increased, and our isotope tracking analysis detected gluconeogenic activities with glycerol most preferred substrate. In addition, many of gluconeogenic genes were expressed at the YSL‐like tissue, suggesting that cloudy catshark engages in gluconeogenesis in this tissue. The gluconeogenesis in teleost YSL and a similar tissue in elasmobranch species implies conserved mechanisms of yolk metabolism between these two lineages. Future studies on other vertebrate taxa will be helpful to understand the evolutionary changes in the modes of yolk metabolism that vertebrates have experienced.

enzymatic reactions where glucose is produced from nonsugar substrates such as amino acids and glycerol, and in vertebrate adult animals the liver and kidney are responsible for this function (Suarez & Mommsen, 1986).Although glucose is considered to play important roles in normal development of brain and hematopoietic stem cells (Harris et al., 2013;Jensen et al., 2006), it is often very scarce in egg yolks (Hamor & Garside, 1977), and the gluconeogenesis in YSL likely contributes to glucose supplementation in the teleost embryos.So far, YSL was believed to induce embryonic differentiation and regulate epiboly in the very early stages of development, and then contribute only to yolk transport and degradation.However, by the above finding, it was inferred that YSL is also responsible for metabolism of yolk components to actively synthesize and supplement the substances in need.Teleost YSL forms during the process of discoidal cleavage, a type of meroblastic cleavage (Kimmel & Law, 1985), and it locates at the most inner layer of yolk sac membrane (YSM) after the end of epiboly.The existence of a tissue structurally similar to teleost YSL (YSL-like tissue) has been reported in the YSM of cloudy catshark and Atlantic sharpnose shark (Rhizoprionodon terraenovae), which also undergo discoidal cleavage (Hamlett & Wourms, 1984;Honda et al., 2022), but its formation process is largely unknown.Thus, whether it has the same function as teleost YSL is unclear.Although the physiological role of YSL-like tissue in cartilaginous fish is unidentified, their yolk contains little glucose (Nishiguchi et al., 2017), similar to teleosts.Hence, we assumed that the YSL-like tissue is involved in the mechanism of yolk utilization to compensate for the lack of glucose.
In the present study, we used cloudy catshark (or Torazame catshark; Scyliorhinus torazame), and examined changes in metabolite levels during their development.Because we confirmed increases in glucose levels in the yolk sac, we incubated the YSM with 13 C-labeled metabolites and tracked their fate by liquid chromatography (LC)/mass spectrometry (MS) analysis.Gene expression analysis was also performed to further corroborate and localize the gluconeogenic activity.The results revealed that the YSM of cloudy catshark undergoes gluconeogenesis, and gene expression profiles suggest that this activity lies in YSL-like tissue, indicating its functional similarity with YSL in teleost fishes.

| Animals
Cloudy catshark (or Torazame catshark) was used in the experiments because it is readily available in coastal areas of Japan (Hara et al., 2018), its genome database is available, and its standard embryonic developmental stages are defined in the closely related species, lesser spotted dogfish (Scyliorhinus canicula; Ballard et al., 1993).Cloudy catsharks were reared in 1000 or 3000-L tanks filled with recirculating seawater in the Atmosphere and Ocean Research Institute, the University of Tokyo.At this site, eggs were routinely laid by mature females and were reared in floating cages in 1000-L tanks under controlled light conditions (12 h light: 12 h dark) at 16°C (for seawater parameters, see Takagi et al., 2017).
In the present study, the embryos and yolk sacs of the following stages were used: stages 4, 24, 27, 31, and 32 (Table S1).The developmental stages of cloudy catshark embryos were identified using a detailed table for lesser spotted dogfish (S. canicula) (Ballard et al., 1993).Every effort was made to minimize the stress to the fish, and the embryos used were anesthetized by exposure to 0.05% tricaine before sampling.These samples were treated as mentioned below for each experiment.

| Metabolite analysis
We measured the following metabolites in the samples by LC/MS analysis: glucose, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), glucose-1-phosphate (G1P), glycogen, glycerol-3-phosphate (G3P), lactate, alanine, threonine, serine, phenylalanine, tyrosine, histidine, proline, glutamine, glutamate, valine, methionine, succinate, and malate.Samples at stages 4, 24, 27, 31, and 32 were used to measure these metabolites (n = 6; Table S1).In stage 24, due to technical reasons (noises in chromatograms), we could only gain clear results of glucose.At sampling, the eggshells were opened by scissors, and the animals were transferred to dishes filled with seawater containing 0.05% tricaine.After the embryos ceased moving, the samples were brought up with a spoon, and the excess water was removed with pieces of paper.Then, the samples were placed in 50-mL centrifuge tubes and weighed.At stages 27, 31, and 32, embryo and yolk sac were separated with scissors after anesthetizing and placed in different tubes.For every 1 g sample, 1 mL of distilled water, 2 mL 100% methanol, and 0.05 mL internal standard [l-methionine sulfone, 2-(N-morpholino) ethanesulfonic acid, d-camphor-10-sulfonic acid, 5 mM each] were added and homogenized.In addition, 2 mL chloroform was added and mixed.After keeping on ice for 10 min, the lysate was centrifuged at 13,000g for 10 min at 4°C, and the aqueous phase was analyzed with an LC system (Prominence; Shimadzu, Kyoto, Japan) connected to a TripleTOF 5600+ mass spectrometer (AB Sciex, Framingham, MA).Shodex HILICpak VG-50 2D (Showa Denko, Tokyo, Japan) was used as the analytical column, and the mobile phase was solvent A: acetonitrile and solvent B: 0.5% aqueous ammonia.The initial concentration was 8.8% B, and the gradient conditions were 8.8% B (8 min), 95% B (14 min), 95% B (17 min), 8.8% B (19 min), and 8.8% B (24 min).The flow rate of the mobile phase was 0.2 mL/min and the column oven temperature was 60°C.For the determination of phosphorylated sugars and amino acids, Shodex HILICpak VT-50 2D (Showa Denko) was used, with solvent A: acetonitrile and solvent B: 25 mM ammonium formate at the constant ratio of A:B = 20:80 as mobile phase.The flow rate of the mobile phase was 0.3 mL per min and the column oven temperature was 60°C.The concentration of each metabolite in the samples was determined with reference to parallel measurement of standard solutions of stepwise dilutions.

| RNA probe synthesis
For in situ hybridization analysis, DIG-labeled RNA probes were prepared.First, target cDNA fragments were amplified with the primers listed in Table 1 and ligated into pGEM T-Easy vector (Promega, Madison, WI).The cDNA fragments were PCR amplified with M13 primers and purified with the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan).The purified cDNA fragments were transcribed in vitro using T7 or SP6 RNA polymerase (Roche Diagnostics) in the presence of digoxigenin (DIG)-UTP.The resulting cRNA probe was digested with DNase I and purified with the RNeasy MinElute cleanup kit (Qiagen, Hilden, Germany).

| In situ hybridization
To examine the localization of target gene transcripts, in situ hybridization analysis was performed according to a previous study (Furukawa et al., 2018).Since adult elasmobranchs undergo gluconeogenesis in the liver (Ballantyne, 2015), this study also looked at gene expression in this organ to compare with YSM.The embryos and YSM at stages 27, 31, and 32 were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline prepared with diethylpyrocarbonate-treated water (PBS-DEPC) overnight at 4°C.Since the embryos of stages 31 and 32 were too large, only the liver was separated from the fixed embryos and further processed for sectioning.The fixed samples were then embedded in paraffin, sectioned into 5-μm slices, and placed onto glass slides.After deparaffinization, sections were digested with 2 μg/mL proteinase K for 30 min and postfixed with 4% PFA in PBS-DEPC for 15 min.The samples were then acetylated with 100 mM triethanolamine (pH 8.0) containing 0.25% acetic anhydride (Hayashi et al., 1978).After prehybridization

| Statistics
All numerical data are expressed as mean ± standard error.The significance of difference at p < 0.05 between the means of groups were tested by one-way analysis of variance (ANOVA) followed by Tukey's and Dunnet's post-hoc test for metabolite analysis and metabolite tracking experiment, respectively.The results of qPCR analysis were analyzed by two-way ANOVA followed by Tukey's test for the significant difference at p < 0.05.To standardize the variations of values among groups, the data were log-normalized before the tests.Statistical analyses were performed with a commercial software (Graphpad Prism 9.31 [350]; GraphPad Software, Boston, MA).

| Metabolite analysis
LC/MS analysis revealed that the levels of many metabolites in the whole embryo and yolk sac (with the yolk contained) of cloudy catshark are highly variable during development (Figure 1; Figure S2).Glucose concentration in the yolk sac increased approximately 100-fold from the egg just after spawning (stage 4) to stage 27, and it sharply increased after stage 24.The levels of glycogen, the storage form of glucose, tended to increase from stage 4 to 24 and then decreased at stage 27.The levels of G6P and F6P, the intermediary metabolites in gluconeogenesis or glycolysis, increased over the development in both the yolk sac and the embryo.In the yolk sac, many amino acids (i.e., threonine, alanine, serine, histidine, glutamine, glutamate, and valine) tended to be richer in stages 27 and 31 than in stages 4 and 32.Except G6P and lactate, all metabolites assessed were incomparably abundant in the yolk sac than in the embryos until stage 31; however, in the embryos these metabolites increased with development, and at stage 32, the levels of G6P, G1P, G3P, lactate, alanine, glutamine, succinate, and malate became comparable to, or surpassed, those in the yolk sac.

| Isotope tracking
Isotope tracing was performed to determine the pathway by which glucose is produced (Jang et al., 2018).Glycerol, lactate, alanine, and glutamate were selected as possible substrates for gluconeogenesis, and the cloudy catshark YSM was incubated in these substrates labeled with 13 C.As a result, levels of glucose with mass + 3 (M + 3) were elevated in the samples incubated with 13 C-labeled glycerol and alanine, which was statistically significant (Figure 2).G6P M + 3 and F6P M + 3/G1P M + 3/sedoheptulose-7phosphate (S7P) M + 3, the intermediates of gluconeogenesis/glycogen metabolism/pentose phosphate pathway from the labeled substrates, were also increased in samples incubated with labeled glycerol, lactate, and alanine, all of which were statistically significant, except G1P M + 3, where only glycerol group reached significance (Figure 2).The results for glucose M + 3 in the samples which took labeled lactate was omitted due to interference of presumably other metabolites.

| Real-time PCR
In Figure 3; Figure S3, expression levels of gluconeogenesisrelated genes and slc2 glucose transporter family were shown.Many of these showed statistically significant differences in expression levels between positions (embryonic body vs. yolk sac; *P) and/or among developmental stages (*S).Significant interactions (*I) between the two factors (developmental stage and position) indicates differential T A B L E 1 (Continued) expression changes between the two positions over developmental stages.Among the gluconeogenic genes, g6pc1, fbp1, pc, pck1, and gys2 were highly expressed in the yolk sac and tended to be most highly expressed at stage 31.In the slc2 glucose transporter gene family, only slc2a2 was highly expressed in the yolk sac and at stage 31.In the embryos, the expression levels of fbp2, gpd1, slc2a5, gys1, and pygm tended to increase over the development, while g6pc3 and slc2a1b showed decreasing tendency.

| In situ hybridization
For the mRNAs detected by real-time PCR, their localization was assessed in YSM and the embryonic livers at stages 27, 31, and 32 by in situ hybridization.In the YSM at stage 27, g6pc1, fbp1, pck2, gpd1, and ldha were expressed in YSL-like tissues and their nuclei (Figure 4).In the same tissues, pck1, gpd1l, and pc were additionally expressed in stage 31 (Figure 5).In stage 32, all transcripts assessed except pck1 were found in YSL-like tissues and yolk sac endoderm (YSE), although the signals of g6pc1 and pc were weak (Figure 6).pck1 was sporadically expressed in YSE at similar intervals.In the liver, transcripts of pck1/pck2, gpd1/gpd1l, pc, and ldha were expressed in stage 27 (Figure S4).In addition to these genes, g6pc1 and fbp1 were expressed in stage 31 and 32 (Figure S5; Figure 6).In stage 31, the signals of g6pc1 were sparse, probably in cells near the sinusoids (Figure S5).These specific mRNA signals generated by antisense probes were not observed in the negative control slides subjected to sense probes.Our metabolite analysis showed that cloudy catshark eggs just after spawning contain very little glucose, but it eventually increased, indicating that glucose is synthesized during the development (Figure 1).In the lesser spotted dogfish, the development from fertilization to hatching is divided into 34 different stages according to the morphological characteristics of the embryo and yolk sac (Ballard et al., 1993).In the present study, glucose content increased as the development proceeded, especially after stage 24 (Figure 1).These changes occurred in the yolk sac samples, implying that YSM is responsible for the increase in glucose content during this period.Although glycogen was degraded in the yolk sac after stage 27, the decrease seemed inadequate to support the glucose production at this time, which led us to assess possible gluconeogenic activity in the YSM.Metabolite tracking in YSM incubated with 13 C-labeled alanine, lactate, or glycerol revealed increases in M + 3 isotopologues of F6P, G6P, G1P, and S7P, the intermediate metabolites for gluconeogenesis, glycogen metabolism, and pentose phosphate pathway (Figure 2).Also, glucose M + 3 was elevated in YSM incubated with labeled alanine or glycerol.These M + 3 isotopologues are most likely those containing three 13 C, which were passed through the gluconeogenic pathway from the substrates added.These results suggest that the labeled substrates are used to produce glucose and glycogen in YSM of the cloudy catshark, which also supports the results of the metabolite analysis.Among the substrates added, glucose was most efficiently produced from glycerol.The yolk of lesser spotted dogfish contains about 20% lipid on dry weight basis (Wrisez et al., 1993), and the total dry mass of the yolk is reduced by about 20% by hatching (Lechenault et al., 1993).On the other hand, approximately 44% of yolk lipids alone are lost, suggesting that these lipids are actively used during development for lipid membrane, energy source, and/or metabolic substrate.Lipids such as triglycerides yield glycerol upon degradation, which may subsequently function as a substrate for gluconeogenesis.These previous studies support the results in the tracer experiments, and taken together, elasmobranchs likely use glycerol as a preferred substrate for gluconeogenesis during development.
F I G U R E 2 LC-MS-based isotope tracking of yolk sac membrane (YSM) in stage 31.Abundance of M + 3 isotopologues of glucose, glucose-6-phosphate (-P), fructose-6-P, glucose-1-P, and sedoheptulose-7-P in the YSM after 3-h incubation with 13 C-labeled substrates.Horizontal axes show each 13 C-labeled substrate and the control without them, and vertical axes show the levels of M + 3 (as % M + 0).Data are presented as mean ± standard error (N = 9), and asterisks (*) indicate significant differences (p < 0.05) between the control and each group.Tests for significant differences were conducted with one-way ANOVA followed by Dunnett's test after log-transformation of the values.N/A, not available.
In our qPCR analysis, many of the gluconeogenesisrelated genes were expressed in the yolk sac (Figure 3), and subsequent in situ hybridization analysis also supported these results.The cloudy catshark YSM consists of: (from the outside) two layers of squamous epithelium, fibrous connective tissue, basement membrane, vascular endothelium, YSE, and YSL-like tissue (Hamlett et al., 1987;Hamlett & Wourms, 1984;Lechenault et al., 1993).Within YSM, many of the gluconeogenesis-related genes were expressed in the YSL-like tissue and/or YSE, even when the signal was absent in the embryonic liver, after stage 31 (Figures 4-6).pck1 was found only in a portion of YSE, implying that YSE consists of more than one type of cells.g6pc1, a gene required to produce glucose from G6P, showed a robust signal in YSL-like tissue at stage 31, while it became weak at stage 32 (Figures 5 and 6), supporting our qPCR data (Figure 3).These results suggest the existence of active gluconeogenesis in the YSL-like tissue at stage 31, and marginal gluconeogenic activity in YSM (YSL-like tissue and YSE) after stage 32.In cloudy catsharks, the anterior portion of the yolk capsule opens during stage 31, a phenomenon called pre-hatching, and at similar timing, yolk reaches the gut through the yolk stalk and starts to be absorbed (Honda et al., 2020).These events trigger increases in the expression of various genes responsible for amino acid transport, lipid absorption, F I G U R E 3 Changes in expression levels of the selected genes during development.The expression levels of gluconeogenesis-related genes and glucose transporter (GLUT) gene slc2a2 were measured.Horizontal and vertical axes indicate developmental stages and mRNA levels (× 10 9 copies/g RNA), respectively.Open and filled columns indicate the mRNA levels in the yolk sac membrane (YSM) and embryos, respectively.Data are presented as mean ± standard error (N = 6), and different letters indicate significant differences (p < 0.05) between groups.Tests for significance were performed by two-way ANOVA and Tukey's post-hoc test.*I indicates a significant interaction between the two factors (developmental stage and site), and *S and *P indicate significant main effects of developmental stage and the position (yolk sac or embryo), respectively.For other genes assessed in this study, please refer to Figure S3.
and digestive enzymes in the gut and yolk sac (Honda et al., 2022).The results of this study suggest that the YSL-like tissue of cloudy catshark actively performs metabolism such as gluconeogenesis using the yolk before the development of many organs.Later, however, its metabolic functions are shifted to the embryo (e.g., liver, intestine, kidney), and YSL-like tissue may focus more on absorption and transport of the yolk together with the gut and YSE (Figure 7).Similar phenomenon was observed in this species for urea synthesis, where this osmolyte was actively synthesized in YSM at early stages, but later the liver takes over this critical physiological function (Takagi et al., 2017).
In the cloudy catshark, many orthologous genes of those expressed in zebrafish YSL were also expressed in YSL-like tissues.The same was true for the slc2 family genes encoding glucose transporters, where slc2a2 was YSL-like type as in zebrafish (Castillo et al., 2009).The functional similarity between zebrafish YSL and cloudy catshark YSL-like tissue in terms of gluconeogenesis may raise a question: do these tissues share a common evolutionary origin, and are they the products of parallel evolution occurring on the same molecular basis (Rosenblum et al., 2014)?However, reports exist that YSL in teleost fishes and shark YSL-like tissues have different nuclear origins.In teleosts, the YSL nuclei are formed by the disintegration of cells at the marginal blastomere (limbal cells) and their fusion with the cytoplasmic layer on the yolk surface (Kimmel & Law, 1985).On the other hand, the elasmobranch YSL-like tissue has primary nuclei derived from the blastoderm and secondary nuclei from the endoderm (Lechenault & Mellinger, 1993).The primary nuclei are believed to be derived from those left behind in the yolk when the segmentation cavity is broken up into yolk and embryo, and it is not clear whether the disintegration of the blastodisc marginal cells occur.Thus, many aspects of the formation, structure, and function of the elasmobranch YSL-like tissues are unknown and may differ from those of teleost YSL.For now it is not possible to determine whether the YSL/YSL-like tissues in these two taxa are the products of parallel evolution or of convergent evolution, the latter occurring on a different molecular basis (Baguñà & Garcia-Fernàndez, 2003).To fill these knowledge gaps, it is necessary to observe and analyze in detail the morphological and molecular aspects of the formation of YSL-like tissues, as well as tracking fates of counterpart cell lineages in other bony fishes and cyclostomes.

| Perspectives and significance
The present study showed that cloudy catsharks undergo gluconeogenesis using glycerol and other substrates in YSM, where the YSL-like tissue likely takes the central role.Gluconeogenesis before organ development is essential and may be a widely conserved phenomenon in many vertebrate animals.Indeed, the YSL of zebrafish undergo gluconeogenesis at the timing of brain formation and blood cell production (Furukawa et al., 2024;Harris et al., 2013;Jensen et al., 2006).Our results suggest that elasmobranch YSL-like tissue also support organogenesis by supplementing glucose: indeed, shark brain express glucose transporter (Balmaceda-Aguilera et al., 2012), implying its dependence on glucose.However, glucose is believed to be less important than ketone bodies as an energy fuel of adult elasmobranchs (Ballantyne, 2015).Since glucose is also consumed to produce nucleic acids or hexosamines via pentose phosphate pathway or hexosamine biosynthetic pathway, respectively, comparing such activities among different ontogenic stages may be interesting in the future.There is still a lot to be uncovered in the yolk metabolism in developing elasmobranchs, and this study provides the important basis for understanding developmental physiology in this group.Since elasmobranchs embryos have many nutritional modes (viviparty etc.), comparing yolk metabolism among such species will be also interesting.Although we have shown a shared function of elasmobranch YSL-like tissue with teleost YSL, it is still unclear whether it was acquired in the common ancestor and modified during evolution to suit each taxon, or whether it was acquired independently.Future studies of the sites of gluconeogenesis in other vertebrate taxa, together with the YSL-like tissue of cloudy catshark, will help us trace the roots of this tissue and to elucidate early developmental changes that vertebrates experienced during their evolution.

F
The metabolic pathway map showing changes in each metabolite levels per individual yolk sac (open column) or embryo (filled column) during development.The horizontal axes represent developmental stages {stages 4, 24 (glucose and glycogen only), 27, 31, 32} and the vertical axes represent nmol/sample.Data are presented as mean ± standard error (N = 6).Different letters indicate significant differences (p < 0.05) between groups.Tests for significant differences were performed by one-way ANOVA and Tukey's post-hoc test separately for yolk sac or embryo samples after log transformation.Fructose-1,6BP, fructose 1,6-bisphosphate; -P, -phosphate.Genes responsible for respective pathways were shown beside the arrows.For the complete map with all metabolites measured in this study, please refer to FigureS2.

F
In situ hybridization analysis for g6pc1, fbp1, pck2, gpd1, ldha, and slc2a2 at yolk sac membrane (YSM) at stage 27.For all transcripts, sense probes (right) were used as negative controls for antisense probes (left), which showed true signals.Positive signals for transcripts are indicated by arrowheads.Hematoxylin and eosin (HE) staining shows general morphology of the YSM in stage 27.se, squamous epithelium; yp, yolk platelet; YSN, yolk syncytial nuclei.Scale bars = 50 μm.

F
Model of functional changes in the yolk sac over the near-prehatching stages.(a) Until stage 31, where pre-hatching takes place, yolk degradation, absorption, and gluconeogenesis occur mainly in the yolk sac; (b) At stage 32 or later, yolk degradation and absorption become more active in both embryonic gut and yolk sac, and glucogenesis and other processes occur actively in the embryo.
List of primers used in this study.
T A B L E 1